EDTA and other chelators with or without antifungal antimicrobial agents for the prevention and treatment of fungal infections

ABSTRACT

A pharmaceutical composition comprising at least one antifungal agent and at least one chelator, and a method for administering the pharmaceutical composition to a patient having a fungal infection. Another aspect provides a pharmaceutical composition comprising at least one chelator, at least one antifungal agent and at least one monoclonal antibody, wherein the monoclonal antibody is operatively attached to the chelator, and a method of administering this composition to a patient having a fungal infection.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/056,970, filed Aug. 26, 1997.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the treatment offungal infections in mammals. More particularly, the present inventionprovides methods of treating fungal infections in mammals usingpharmaceutical preparations including chelator(s), antifungal agents,and/or monoclonal antibodies. The invention further providespharmaceutical compositions useful for treating fungal infections.

[0004] 2. Description of Related Art

[0005] Fungi, particularly species of Candida, Aspergillus, and Fusariumare a major cause of infection-related mortality in patients withleukemia and lymphoma. In addition, fungal infection is a major cause ofmortality in patients with congenital and acquired deficiencies of theimmune system.

[0006] For example, several species of Aspergillus are known to causeinvasive sinopulmonary infections in seriously immunocompromisedpatients. Following inhalation of spores, clinical aspergillosis canoccur in three major presentations. The first presentation, allergicbronchopulmonary aspergillosis, develops when Aspergillus speciescolonize the bronchial tree and release antigens that cause ahypersensitivity pneumonitis. The second presentation, aspergilloma or“fungus ball,” develops in pulmonary cavities, often in concert withother lung diseases such as tuberculosis. The third form, invasivepulmonary or disseminated aspergillosis, is a life threatening infectionwith a high mortality rate.

[0007] The drug of choice in treatment of invasive aspergillosis, aswell as in most other systemic mycoses, is Amphotericin B. AmphotericinB is a polyene antibiotic produced from a strain of Streptomycesnodosus. It is a lipophilic compound which binds to ergosterols infungal membranes, resulting in the formation of transmembrane channelswhich allow the escape of metabolites essential to maintaining theviability of the fungal cell. Mammalian cell membranes also containsterols, and it is believed that this same mechanism of action isresponsible for the damaging effects which Amphotericin B is known toexert on mammalian kidney, hematopoietic and central nervous systemtissues.

[0008] Amphotericin B is not soluble in aqueous solution, and for thisreason it is supplied commercially in the form of a colloidal suspensioncomprising Amphotericin B, desoxycholate, and buffers suspended in aglucose solution. This suspension is usually administered to the patientintravenously over a period of from two to six hours; faster infusionscan result in cardiorespiratory arrest. Other possible untoward effectsof administering Amphotericin B include fever, nausea and vomiting,diarrhea, renal dysfunction, anemia, hypotension, headache, vertigo, andloss of hearing. Amphotericin B is also available in the form of aphospholipid complex (ABELCET®, e.g.), which offers the advantage ofsomewhat reduced toxicity for those patients who do not tolerate freeAmphotericin B well, although many of the same untoward side effects maybe observed in patients receiving this lipid complex form of the drug.

[0009] As a consequence of the potential seriousness of its toxic sideeffects, there is a clear need for an alternative to treating systemicmycoses solely with Amphotericin B and/or other harsh antifungal agents.

SUMMARY OF THE INVENTION

[0010] The present invention provides an effective method of treating asystemic fungal infection comprising the steps of obtaining atherapeutically effective amount of a pharmaceutical compositioncomprising at least one chelator, at least one antifungal agent and apharmaceutical excipient, diluent or adjuvant, and administering saidpharmaceutical composition to a patient having a fungal infection.

[0011] For the purposes of this disclosure, the phrase “therapeuticallyeffective amount” is defined as a dosage sufficient to induce afungicidal or fungistatic effect upon fungi contacted by thecomposition. That amount of the pharmaceutical composition which istherapeutically effective will depend upon the ingredients comprisingthe composition, as well as the treatment goals.

[0012] For the purposes of this disclosure, the phrase “a chelator”denotes one or more chelators. As used herein, the term “chelator” isdefined as a molecule comprising nonmetal atoms, two or more of whichatoms are capable of linking or binding with a metal ion to form aheterocyclic ring including the metal ion.

[0013] For the purposes of this disclosure, the phrase “an antifungalagent” denotes one or more antifungal agents. As used herein, the term“antifungal agent” is defined as a compound having either a fungicidalor fungistatic effect upon fungi contacted by the compound.

[0014] As used herein, the term “fungicidal” is defined to mean having adestructive killing action upon fungi. As used herein, the term“fungistatic” is defined to mean having an inhibiting action upon thegrowth of fungi.

[0015] As used herein the terms “contact”, “contacted”, and“contacting”, are used to describe the process by which apharmacological agent, e.g., any of the compositions disclosed in thepresent invention, comes in direct juxtaposition with the target cell.

[0016] Preferable chelators for use in the present invention include,but are not limited to, ethylenediamine-N,N,N′,N′-tetraacetic acid(EDTA); the disodium, trisodium, tetrasodium, dipotassium, tripotassium,dilithium and diammonium salts of EDTA; the barium, calcium, cobalt,copper, dysprosium, europium, iron, indium, lanthanum, magnesium,manganese, nickel, samarium, strontium, and zinc chelates of EDTA;trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraaceticacid monohydrate;N,N-bis(2-hydroxyethyl)glycine;1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid;1,3-diaminopropane-N,N,N′,N′-tetraacetic acid;ethylenediamine-N,N′-diacetic acid; ethylenediamine-N,N′-dipropionicacid dihydrochloride; ethylenediamine-N,N′-bis(methylenephosphonic acid)hemihydrate; N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid;ethylenediamine-N,N,N′,N′-tetrakis(methylenephosponic acid);O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid;N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid;1,6-hexamethylenediamine-N,N,N′,N′-tetraacetic acid;N-(2-hydroxyethyl)iminodiacetic acid; iminodiacetic acid;1,2-diaminopropane-N,N,N′,N′-tetraacetic acid; nitrilotriacetic acid;nitrilotripropionic acid; the trisodium salt ofnitrilotris(methylenephosphoric acid);7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo [11,11,11]pentatriacontane hexahydrobromide; andtriethylenetetramine-N,N,N′,N″,N′″,N′″-hexaacetic acid. It iscontemplated that any chelator which binds barium, calcium, cerium,cobalt, copper, iron, magnesium, manganese, nickel, strontium, or zincwill be acceptable for use in the present invention.

[0017] More preferably, the chelators for use in conjunction with thepresent invention may include ethylenediamine-N,N,N′,N′-tetraacetic acid(EDTA); the disodium, trisodium, tetrasodium, dipotassium, tripotassium,dilithium and diammonium salts of EDTA;1,3-diamino-2-hydroxypropane-N,N,N′,N′-tetraacetic acid;1,3-diaminopropane-N,N,N′,N′-tetraacetic acid;O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid; and7,19,30-trioxa-1,4,10,13,16,22,27,33-octaazabicyclo [11,11,11]pentatriacontane hexahydrobromide.

[0018] Most preferably, the chelators for use in the present inventionmay include ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA); thedisodium salt of EDTA; 1,3-diaminopropane-N,N,N′,N′-tetraacetic acid;and O,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid.

[0019] Many antifungal agents are known to those of skill in the art andmay be useful in the present invention. For example, antifungal agentscontemplated for use in the present invention include, but are notlimited to, new third generation triazoles such as UK 109,496,(Voriconazole); SCH 56592; ER30346; UK 9746; UK 9751; T 8581; andFlutrimazole; cell wall active cyclic lipopeptides such as CilofunginLY121019; LY303366 (Echinocandin); and L-743872 (Pneumocandin);allylamines such as Terbinafine; imidazoles such as Omoconazole,Ketoconazole, Terconazole, Econazole, Itraconazole and Fluconazole;polyenes such as Amphotericin B, Nystatin, Natamycin, LiposomalAmphotericin B, and Liposomal Nystatin; and other antifungal agentsincluding Griseofulvin; BF-796; MTCH 24; BTG-137586; RMP-7/AmphotericinB; Pradimicins (MNS 18184); Benanomicin; Ambisome; ABLC; ABCD;Nikkomycin Z; and Flucytosine.

[0020] More preferably, the antifungal agents for use in conjunctionwith the present invention may include polyenes such as Amphotericin B,Nystatin, Natamycin, Liposomal Amphotericin B, and Liposomal Nystatin;cell wall active cyclic lipopeptides such as Cilofungin LY121019;LY303366 (Echinocandin); and L-743872 (Pneumocandin); and otherantifungal agents including Griseofulvin and Flucytosine.

[0021] Most preferably, the antifungal agents for use in the presentinvention may include Amphotericin B, Nystatin, Liposomal AmphotericinB, and Liposomal Nystatin.

[0022] The present invention also provides an effective method oftreating a systemic fungal infection comprising the steps of obtaining atherapeutically effective amount of a pharmaceutical compositioncomprising at least one chelator operatively attached to a monoclonalantibody, at least one antifungal agent and a pharmaceutical excipient,diluent or adjuvant, and administering said pharmaceutical compositionto a patient having a fungal infection. The monoclonal antibody ischosen to bind to a specific fungal antigen, and may be preparedaccording to any known method. The chelators and antifungal agents maybe chosen from those indicated above.

[0023] Monoclonal antibodies useful in conjunction with the presentinvention are those that are specific for a targeted species of fungus.In preferred embodiments, the monoclonal antibodies are operativelyattached to chelators. For the purposes of this disclosure, the phrase“a monoclonal antibody” denotes one or more monoclonal antibodies. Asused herein, the term “monoclonal antibody” is defined as an antibodyderived from a single clone of a B lymphocyte. Furthermore, as usedherein, the term “operatively attached” connotes a chemical bond, eithercovalent or ionic, between the monoclonal antibody and chelator. As usedherein, the term “specific” indicates that a chemical site on themonoclonal antibody will recognize and bind with a complementarychemical site on the surface of the cell of at least the fungal pathogenof interest.

[0024] The pharmaceutical compositions of the invention are provided toa patient having a fungal infection in an amount sufficient to exert afungicidal or fungistatic effect upon fungi contacted by thecomposition. It will be understood with benefit of this disclosure thatsuch dosages may vary considerably according to the patient, theinfection presented by the patient, and the particular activeingredients comprising the pharmaceutical composition.

[0025] The antifungal agents of the present invention may beadministered to a patient in an amount ranging from about 0.001milligrams per kilogram of body weight per day to about 1000 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.001, 0.002,0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10,etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100, 101, 102, 103,104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800, 900, and about 1000mg per kg per day, and including all fractional dosages therebetween.

[0026] More preferably, the antifungal agents of the present inventionmay be administered to a patient in an amount ranging from about 0.01milligrams per kilogram of body weight per day to about 100 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.01, 0.02, 0.03,etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, andincluding all fractional dosages therebetween.

[0027] Most preferably, the antifungal agents of the present inventionmay be administered to a patient in an amount ranging from about 0.1milligrams per kilogram of body weight per day to about 10 mg per kg perday, including all intermediate dosages therebetween. It will be readilyunderstood that “intermediate dosages”, in these contexts, means anydosages between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,7, 8, 9 and about 10 mg per kg per day, and including all fractionaldosages therebetween.

[0028] The chelators of the present invention may be administered to apatient in an amount ranging from about 0.001 milligrams per kilogram ofbody weight per day to about 1000 mg per kg per day, including allintermediate dosages therebetween. It will be readily understood that“intermediate dosages”, in these contexts, means any dosages between thequoted ranges, such as about 0.001, 0.002, 0.003, etc.; 0.01, 0.02,0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.;50, 51, 52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day, andincluding all fractional dosages therebetween.

[0029] More preferably the chelators of the present invention may beadministered to a patient in an amount ranging from about 0.01milligrams per kilogram of body weight per day to about 100 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.01, 0.02, 0.03,etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, andincluding all fractional dosages therebetween.

[0030] Most preferably the chelators of the present invention may beadministered to a patient in an amount ranging from about 0.1 milligramsper kilogram of body weight per day to about 10 mg per kg per day,including all intermediate dosages therebetween. It will be readilyunderstood that “intermediate dosages”, in these contexts, means anydosages between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,7, 8, 9, and about 10 mg per kg per day, and including all fractionaldosages therebetween.

[0031] The monoclonal antibodies operatively attached to chelators maybe administered to a patient in an amount ranging from about 0.001milligrams per kilogram of body weight per day to about 1000 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.001, 0.002,0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10,etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100, 101, 102, 103,104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800, 900, and about 1000mg per kg per day, and including all fractional dosages therebetween.

[0032] More preferably, the monoclonal antibodies operatively attachedto chelators may be administered to a patient in an amount ranging fromabout 0.01 milligrams per kilogram of body weight per day to about 100mg per kg per day, including all intermediate dosages therebetween. Itwill be readily understood that “intermediate dosages”, in thesecontexts, means any dosages between the quoted ranges, such as about0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12,13, 14, etc.; 50, 51, 52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mgper kg per day, and including all fractional dosages therebetween.

[0033] Most preferably, the monoclonal antibodies operatively attachedto chelators may be administered to a patient in an amount ranging fromabout 0.1 milligrams per kilogram of body weight per day to about 10 mgper kg per day, including all intermediate dosages therebetween. It willbe readily understood that “intermediate dosages”, in these contexts,means any dosages between the quoted ranges, such as about 0.1. 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.;3, 4, 5, 6, 7, 8, 9, and about 10, and including all fractional dosagestherebetween.

[0034] The pharmaceutical compositions of the present invention may beadministered by any known route, including parenterally and otherwise.This includes oral, nasal (via nasal spray or nasal inhaler), buccal,rectal, vaginal or topical administration. Administration may also be byorthotopic, intradermal subcutaneous, intramuscular, intraperitoneal orintravenous injection and/or infusion. Such compositions may beadministered as pharmaceutically acceptable compositions that includepharmacologically acceptable carriers, buffers or other excipients. Thephrase “pharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to a human. For treatment of conditions ofthe lungs, the preferred route is aerosol delivery to the lung viabronchoalveolar lavage or the like.

[0035] When administration of the pharmaceutical compositions of thepresent invention via intravenous injection and/or infusion is thepreferred route, the pharmaceutical compositions of the presentinvention should administered gradually over a period of time rangingfrom 0.001 h to 100 h. More preferably, when administration of thepharmaceutical compositions of the present invention via intravenousinjection and/or infusion is the preferred route, the pharmaceuticalcompositions of the present invention should administered gradually overa period of time ranging from 0.1 h to 50 h. Most preferably, whenadministration of the pharmaceutical compositions of the presentinvention via intravenous injection and/or infusion is the preferredroute, the pharmaceutical compositions of the present invention shouldadministered gradually over a period of time ranging from 1 h to 10 h.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein. FIG. 1, FIG. 2, FIG. 3, FIG. 4,FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11 displayplots of microbial population vs. time for cultures of species ofAspergillus, Candida and Fusarium. Response of these cultures totreatment with antimicrobials, chelators, and combinations thereof areindicated.

[0037]FIG. 1 shows the inhibitory effect of EDTA on Aspergillus flavusin vitro.

[0038]FIG. 2 shows the inhibitory effect of EDTA on Aspergillus terreusin vitro.

[0039]FIG. 3 shows the inhibitory effect of EDTA on Fusarium oxysporumin vitro.

[0040]FIG. 4 shows the inhibitory effect of EDTA on Candida krusei invitro.

[0041]FIG. 5 shows the synergistic inhibition of Aspergillus fumigatusby Amphotericin B and EDTA (1.0 mg/mL) in vitro.

[0042]FIG. 6 shows the synergistic inhibition of Aspergillus fumigatusby Amphotericin B and EDTA (0.1 mg/mL) in vitro.

[0043]FIG. 7 shows the synergistic inhibition of Aspergillus flavus byAmphotericin B and EDTA (1.0 mg/mL) in vitro.

[0044]FIG. 8 shows the synergistic inhibition of Aspergillus flavus byAmphotericin B and EDTA (0.1 mg/mL) in vitro.

[0045]FIG. 9 shows the synergistic inhibition of Fusarium solani byAmphotericin B and EDTA in vitro.

[0046]FIG. 10 shows the synergistic inhibition of Aspergillus fumigatusby Ambisome and EDTA (0.1 mg/mL) in vitro.

[0047]FIG. 11 shows the synergistic inhibition of Fusarium solani byAmbisome and EDTA in vitro.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0048] I. Fungal Infections

[0049] Infection is believed to be the cause of death in almost half ofall patients who die of lymphoma, and in almost three quarters ofpatients who die of leukemia. Although bacteria are the causativeorganisms of many such infections, fungi also play a major role in theseinfection-related mortalities. As noted in the Background section,species of Candida, Aspergillus, and Fusarium are the major causes offungal infection-related deaths in patients with leukemia and lymphoma.Additionally, fungal infection is a major cause of mortality in patientswith congenital and acquired deficiencies of the immune system.

[0050] In their usual role as saprophytes on decaying vegetation,Aspergillus organisms are not contagious. Infection arises when sporesof these ubiquitous fungi are aerosolized in the abiotic environment. Nounderlying predisposition of race, age, or sex leads to the developmentof Aspergillus infection.

[0051] Although Aspergillus infection can be acquired in the community,the largest threat to the immunocompromised patient is exposure tocontaminated air in the hospital environment. Patients who haveundergone open heart surgery, who have undergone organ or bone marrowtransplantation, or who have prolonged neutropenia after anticancerchemotherapy may acquire life-threatening infection either on the wardswhere they are housed (domiciliary exposure) or when they are taken tothe operating theater, radiology suite, or catheterization laboratoryfor an essential procedure (nondomiciliary exposure). Construction inthe hospital, which results in the liberation of large numbers of sporesinto the immediate environment, is particularly hazardous for thesepatients (Rubin, 1994; Wade, 1994). Although there is considerablegenetic heterogeneity among Aspergillus strains in nature, newlydeveloped molecular typing systems have shown that patients are usuallyinfected with a single strain, a finding that allows the clinician torapidly assess whether two or more cases are from the same environmentalsource (Birch et al., 1995).

[0052] Several species of Aspergillus are known to cause invasivesinopulmonary infections in seriously immunocompromised patients.Following inhalation of spores, clinical aspergillosis can occur inthree major presentations. The first presentation, allergicbronchopulmonary aspergillosis, develops when Aspergillus speciescolonize the bronchial tree and release antigens that cause ahypersensitivity pneumonitis. The second presentation, aspergilloma or“fungus ball,” develops in pulmonary cavities, often in concert withother lung diseases such as tuberculosis. The third form, invasivepulmonary or disseminated aspergillosis, is a life threatening infectionwith a very high mortality rate.

[0053] The present invention provides pharmaceutical compositions andmethods for the prevention and treatment of disseminated fungalinfections. It is contemplated that the preparations of the inventionwill be useful in eliminating or inhibiting all types of fungalinfections, providing so-called fungicidal or fungistatic effects. Forexample, the inventors have discovered that chelators have significantgrowth inhibitory effect against species of Aspergillus (see data inFIG. 1, FIG. 2, FIG. 3 and FIG. 4). The inventors have furtherdemonstrated conclusively and unexpectedly that, when combined withantifungal agents, chelators show additive to synergistic inhibitoryactivity against the growth of fungal pathogens (see data in FIG. 5,FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11). These discoveriesprovide the basis for a program of prevention and treatment of systemicfungal infections using any of several embodiments of the inventivepharmaceutical formulations, which may comprise various combinations ofchelators, antifungal agents, and any necessary excipients, diluents oradjuvants.

[0054] In another aspect, the pharmaceutical formulations providedherein comprise chelators chemically bound, covalently, ionically orotherwise, to monoclonal antibodies which bind to the fungal antigen tobe treated. Advantageously, the monoclonal antibodies may serve todeliver the attached chelators directly to their intended fungaltargets, where the chelators may then bind trace metals such as iron andcalcium which might otherwise serve as fungal virulence factors. Theinventors contemplate that this preferred embodiment even moreeffectively eliminates or inhibits fungal infections by decreasing thetendency of the chelators to bind trace metals with which they may comeinto contact outside of the immediate vicinity of the infection

[0055] II. Chelators

[0056] A chelate is the type of coordination compound in which a centralmetal ion is attached by coordinate links to two or more nonmetal atomsin the same molecule. Heterocyclic rings are thus formed duringchelation, with the metal atom as part of the ring. The moleculecomprising the nonmetal linking atoms is termed a chelator. Chelatorsare used in various chemical applications, for example as titratingagents or as metal ion scavengers. Chelators can be used to remove ionsfrom participation in biological reactions. For example, the well-knownchelator ethylenediamine-N,N,N′,N′,-tetraacetic acid (EDTA) acts as ananticoagulant because it is capable of scavenging calcium ions from theblood.

[0057] It is known that iron and other trace metals are essential in thelife cycle of microorganisms such as fungi. Without these trace metals,fungi are unable to grow and reproduce. Although iron is abundant innature, its availability for microbial assimilation is limited owing tothe insolubility of ferric ions at neutral or alkaline pH. As aconsequence, many fungi have evolved their own specialized tracemetal-scavenging molecules, called siderophores, which bind with tracemetals and make them available for uptake by the fungi. The chelatorsused in conjunction with the present invention provide an inhibitoryeffect upon fungal pathogens by competing with the fungal siderophoresfor any available trace metal ions. In this way, the chelators presentin the pharmaceutical preparations of the invention “steal” the metalions essential for fungal growth, effectively causing the fungus to“starve to death.” The added antifungal agents and/or monoclonalantibodies of the preparations of the invention can then come in andattack the weakened fungi, thereby destroying them or inhibiting theirgrowth.

[0058] The inventors have discovered that the chelators of the presentinvention have significant growth inhibitory effect against species ofAspergillus. Referring to FIG. 1, it will be seen that EDTA exerts aninhibitory effect upon Aspergillus flavus relative to the controlpopulation. This effect is most clearly noticeable beginning 12 h afterapplication of the chelator. Referring to FIG. 2 and FIG. 3, similarinhibitory behavior was noticed in cultures of Aspergillus terreus andFusarium oxysporum following application of EDTA. The inhibitory effectof EDTA on Candida krusei is noticeable only a few hours after contactof the fungus with the chelator, as shown by FIG. 4.

[0059] Table 1 provides a representative list of chelators useful inconjunction with the present invention. The list provided in Table 1 isnot meant to be exhaustive. Preferred chelators are those which bindtrace metal ions with a binding constant ranging from 10¹ to 10¹⁰⁰; morepreferred chelators are those which bind trace metal ions with a bindingconstant ranging from 10¹⁰ to 10⁸⁰; most preferred chelators are thosewhich bind trace metal ions with a binding constant ranging from 10¹⁵ to10⁶⁰. Also, preferred chelators are those which are readily attached toa monoclonal antibody, for example1,3-diaminopropane-N,N,N′,N′-tetraacetic acid (DTPA). Furthermore,preferred chelators are those which have been shown to have aninhibitory effect upon target fungal pathogens, for example the disodiumsalt of EDTA. TABLE 1 CHELATORS ABBREVIATION FULL NAME EDTA free acidEthylenediamine-N,N,N′,N′,-tetraacetic acid EDTA 2NaEthylenediamine-N,N,N′,N′,-tetraacetic acid, disodium salt, dihydrateEDTA 3Na Ethylenediamine-N,N,N′,N′,-tetraacetic acid, trisodium salt,trihydrate EDTA 4Na Ethylenediamine-N,N,N′,N′-tetraacetic acid,tetrasodium salt, tetrahydrate EDTA 2KEthylenefisminr-N,N,N′,N′-tetraacetic acid, dipotassium salt, dihydrateEDTA 2Li Ethylenediamine-N,N,N′,N′-tetraacetic acid, dilithium salt,monhydrate EDTA 2NH₄ Ethylenediamine-N,N,N′,N′-tetraacetic acid,diammonium salt EDTA 3K Ethylenediamine-N,N,N′,N′-tetraacetic acid,tripotassium salt, dihydrate Ba(II) -EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, barium chelate Ca(II) -EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, calcium chelate Ce(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, cerium chelate Co(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, cobalt chelate Cu(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, copper chelate Dy(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, dysprosium chelateEu(III) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, europiumchelate Fe(III) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, ironchelate In(III) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, indiumchelate La(III) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid,lanthanum chelate Mg(II) -EDTA Ethylenediamine-N,N,N′,N′-tetraaceticacid, magnesium chelate Mn(II) -EDTAEthylenediamine-N,N,N′,N′-tetraacetic acid, manganese chelate Ni(II)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, nickel chelate Sm(III)-EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, samarium chelateSr(II) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, strontiumchelate Zn(II) -EDTA Ethylenediamine-N,N,N′,N′-tetraacetic acid, zincchelate CyDTA trans-1,2-Diaminocyclohexane-N,N,N′,N′- tetraaceticacidmonohydrate DHEG N,N-Bis(2-hydroxyethyl)glycine DTPA-OH1,3-Diamino-2-hydroxypropane-N,N,N′,N′- tetraacetic acid DTPA1,3-Diaminopropane-N,N,N′,N′- tetraacetic acid EDDAEthylenediamine-N,N′-diacetic acid EDDP Ethylenediamine-N,N′-dipropionicacid dihydrochloride EDDPO Ethylenediamine-N,N′-bis (methylenephosphonicacid), hemihydrate EDTA-OH N-(2-Hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid EDTPO Ethylenediamine-N,N,N′,N′-tetrakis(methylenephosponic acid) EGTAO,O′-bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′- tetraacetic acid HBEDN,N-bis(2-hydroxybenzyl)ethylenediamine-N,N- diacetic acid HDTA1,6-Hexamethylenediamine-N,N,N′,N′- tetraacetic acid HIDAN-(2-Hydroxyethyl)iminodiacetic acid IDA Iminodiacetic acid Methyl-EDTA1,2-Diaminopropane-N,N,N′,N′-tetraacetic acid NTA Nitrilotriacetic acidNTP Nitrilotripropionic acid NTPO Nitrilotris(methylenephosphoric acid),trisodium salt O-Bistren 7,19,30-Trioxa-1,4,10,13,16,22,27,33-octaazabicyclo [11,11,11] pentatriacontane, hexahydrobromide TTHATriethylenetetramine - N,N,N′,N″,N′′′,N′′′- hexaacetic acid

[0060] III. Monoclonal Antibodies

[0061] Monoclonal antibodies are antibodies derived from a single cloneof B lymphocytes. As such, they are homogenous in structure and antigenspecificity, making them useful as vectors for directing radionuclides,drugs, or toxins to tissues of interest such as malignant cells.

[0062] In one embodiment, the pharmaceutical formulations of theinvention may comprise chelators chemically attached to monoclonalantibodies which have been designed to bind to the fungal antigen siteof interest. Advantageously, the monoclonal antibodies may serve todeliver the attached chelators directly to their intended fungaltargets, where the chelators may then bind trace metals which exist inthe vicinity of the fungal cell. By delivering the chelator directly toits intended target, scavenging of any nearby trace metals which mightotherwise serve as fungal virulence factors is assured.

[0063] Means for preparing and characterizing antibodies are well knownin the art (See, e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988; incorporated herein by reference). The methodsfor generating monoclonal antibodies (MAbs) generally begin along thesame lines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of anti-antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

[0064] As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-diazotized benzidine.

[0065] As is also well known in the art, the immunogenicity of aparticular immunogen composition can be enhanced by the use ofnon-specific stimulators of the immune response, known as adjuvants.Exemplary and preferred adjuvants include complete Freund's adjuvant (anon-specific stimulator of the immune response containing killedMycobacterium tuberculosis), incomplete Freund's adjuvants and aluminumhydroxide adjuvant.

[0066] The amount of immunogen composition used in the production ofpolyclonal antibodies varies upon the nature of the immunogen as well asthe animal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). The production of polyclonalantibodies may be monitored by sampling blood of the immunized animal atvarious points following immunization. A second, booster injection, mayalso be given. The process of boosting and titering is repeated until asuitable titer is achieved. When a desired level of immunogenicity isobtained, the immunized animal can be bled and the serum isolated andstored, and/or the animal can be used to generate MAbs.

[0067] MAbs may be readily prepared through use of well-knowntechniques, such as those exemplified in U.S. Pat. No. 4,196,265,incorporated herein by reference. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., a purified or partially purified administration of approximately10⁶ Aspergillus fumigatus conidia (or other species, such as Aspergillusflavus, Aspergillus terreus, Candida krusei, or Fusarium solani, or anyother fungal pathogen known to affect humans) in 1.0 mL of sterile NaClsolution with 1.0 mL of Freund's incomplete adjuvant. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, 1986, pp. 60-61), butmice are preferred, with the BALB/c mouse being most preferred as thisis most routinely used and generally gives a higher percentage of stablefusions.

[0068] Following immunization, somatic cells with the potential forproducing antibodies, specifically B lymphocytes (B cells), are selectedfor use in the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

[0069] The antibody-producing B lymphocytes from the immunized animalare then fused with cells of an immortal myeloma cell, generally one ofthe same species as the animal that was immunized. Myeloma cell linessuited for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

[0070] Any one of a number of myeloma cells may be used, as are known tothose of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM 1500-GRG2, LICR-LON-HMy2and UC729-6 are all useful in connection with human cell fusions.

[0071] One preferred murine myeloma cell is the NS-1 myeloma cell line(also termed P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

[0072] Methods for generating hybrids of antibody-producing spleen orlymph node cells and myeloma cells usually comprise mixing somatic cellswith myeloma cells in a 2:1 proportion, though the proportion may varyfrom about 20:1 to about 1:1, respectively, in the presence of an agentor agents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

[0073] Fusion procedures usually produce viable hybrids at lowfrequencies, about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose aproblem, as the viable, fused hybrids are differentiated from theparental, unfused cells (particularly the unfused myeloma cells thatwould normally continue to divide indefinitely) by culturing in aselective medium. The selective medium is generally one that contains anagent that blocks the de novo synthesis of nucleotides in the tissueculture media. Exemplary and preferred agents are aminopterin,methotrexate, and azaserine. Aminopterin and methotrexate block de novosynthesis of both purines and pyrimidines, whereas azaserine blocks onlypurine synthesis. Where aminopterin or methotrexate is used, the mediais supplemented with hypoxanthine and thymidine as a source ofnucleotides (HAT medium). Where azaserine is used, the media issupplemented with hypoxanthine.

[0074] The preferred selection medium is HAT. Only cells capable ofoperating nucleotide salvage pathways are able to survive in HAT medium.The myeloma cells are defective in key enzymes of the salvage pathway,e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannotsurvive. The B cells can operate this pathway, but they have a limitedlife span in culture and generally die within about two weeks.Therefore, the only cells that can survive in the selective media arethose hybrids formed from myeloma and B cells.

[0075] This culturing provides a population of hybridomas from whichspecific hybridomas are selected. Typically, selection of hybridomas isperformed by culturing the cells by single-clone dilution in microtiterplates, followed by testing the individual clonal supernatants (afterabout two to three weeks) for the desired reactivity. The assay shouldbe sensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

[0076] The selected hybridomas would then be serially diluted and clonedinto individual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

[0077] The inventors also contemplate the use of a molecular cloningapproach to generate monoclonals. For this, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies.

[0078] IV. Attachment of Chelators to Monoclonal Antibodies

[0079] Methods for attachment of chelators to proteins, includingmonoclonal antibodies, are well known in the art. One of the earliestefforts at attachment of metal-binding groups to proteins was that ofGelewitz et al. (1954) who coupled azo-phenanthroline and azo-oxine toalbumin; attempts were made to analyze the products by colorimetrictitration with iron. Later, Sokolovsky et al., (1967) converted lysozymetyrosine to 3-aminotyrosine, and discussed the potential of thisprocedure to yield new heavy-atom derivatives for x-ray crystallography.Benisek and Richards (1968) explored the use of picolinimidate toproduce metal-binding sites on proteins, with a similar goal.

[0080] The first chelate-protein conjugates that were stable enough foruse in vivo were the protein azophenyl-EDTAs of Sundberg et al. (1974)which resulted from the initial ideas of Baldeschwieler [Meares et al.(1984)]. Those protein azophenyl-EDTA compounds were studied over theyears 1974-1979, during which the basic requirements of protein-chelatedirected toward in vivo use were explored (Goodwin et al., 1975; Meareset al., 1976; Leung et al., 1978; DeRiemer et al., 1979).

[0081] In 1976, Krejcarek and Tucker activated DTPA by a mixed anhydridemethod and coupled it to albumin (Krejcarek and Tucker, 1976). Theproduct bound ¹¹¹In stably enough for many applications in vivo, and hasbeen used by many nuclear medicine researchers since (Khaw et al., 1980;Scheinberg, 1982). The procedure was subsequently improved by Hnatowichet al. (1983). Comparison of the stability of ¹¹¹In complexes in humanserum under physiological conditions shows that the indium is lost fromDTPA complexes much more rapidly than from phenyl-EDTA complexes,whether bound to a protein or not (Yeh et al., 1979). However,¹¹¹In-DTPA complexes decompose slowly enough so that they are useful inmany diagnostic procedures, including those involving antibodies.

[0082] By 1979, a general method for converting a-amino acids tobifunctional chelating agents had been devised (Yeh et al., 1979). Thishas permitted the synthesis of a wide range of structures from materialswith an interesting choice of useful sidechains.

[0083] Typically, an amino acid such as L-phenylalanine is nitrated,esterified, and allowed to react with an amine RNH₂. If R═H, the finalproduct will be an EDTA analog, whereas if R═H₂NCH₂CH₂—, the finalproduct will be a DTPA analog. The amide is reduced, and the amines arecarboxymethylated to form a chelating group, and then the aromatic nitrogroup is reduced to an amine. This aromatic amine can be furthermodified in several ways to form useful derivatives. For example,treatment with nitric acid renders a diazonium compound which may reactwith several different amino acid residues.

[0084] As noted above, in embodiments of the invention where thechelator is operatively attached to a monoclonal antibody, it iscontemplated that such attachment may be by either covalent or ionicbond. One example of such an attachment is the diazophenyl couplingdescribed above, but any other means of chemically binding a chelator toa monoclonal antibody may be used.

[0085] V. Antifungal Agents

[0086] Fungal infections and the drugs used to treat them havetraditionally been divided into two classes: superficial and systemic.This distinction is becoming increasingly arbitrary, however, as some ofthe drugs previously used to treat only one class of infection or theother are now used in both cases, with the differences being ones ofmode of administration and/or concentration of active ingredient. Also,some infections, for example superficial mycoses, may now be treatedeither systemically or topically.

[0087] The classes of drugs used currently to treat systemic fungalinfections include the polyenes, the imidazoles and triazoles,griseofulvin, and flucytosine. The polyenes bind to ergosterols infungal membranes, resulting in the formation of transmembrane channelswhich allow the escape of metabolites essential to maintaining theviability of the fungal cell. Polyenes are highly toxic. The imidazolesand triazoles are structurally related and share the same antifungalspectrum and mechanism of action, namely the inhibition of the fungalsterol 14-α-demethylase enzyme system. Griseofulvin was isolated from aspecies of Penicillium and acts by inhibiting fungal mitosis.Flucytosine is a fluorinated pyrimidine which acts upon fungi byinhibiting thymidylate synthetase.

[0088] In addition to the above well-known classes of antifungal agents,some compounds more traditionally thought of as antibacterial agents,for example minocycline (a tetracycline derivative), have beendemonstrated to have a fungistatic or fungicidal effect on the surfaceof a venous catheter when administered in combination with a chelator,for example EDTA, and other compounds. See, for example, U.S. Pat. No.5,362,754 by Raad et al., or U.S. patent application Ser. No. 08/317,309by Raad et al., both of which are herein incorporated by reference.

[0089] Antifungal agents particularly preferred in connection with thepresent invention include the polyenes, most preferably Amphotericin Band liposomal Amphotericin B. The inventors have demonstrated thatAmphotericin B acts synergistically in concert with the chelator EDTA toinhibit species of Aspergillus and Fusarium. A drug combination is saidto exhibit synergism when the combination achieves a desired effect oneorder of magnitude or greater than the analogous effect of the mostpotent individual constituent of the combination. For example, referringto FIG. 5, Amphotericin B at a concentration of 1 μg/mL and EDTA at aconcentration of 1 mg/mL act synergistically to inhibit the growth ofAspergillus fumigatus by a margin of almost two orders of magnituderelative to EDTA acting alone. The same effect is observed when theconcentration of EDTA is reduced to 0.1 mg/mL (FIG. 6). Likewise,Amphotericin B and EDTA inhibit Aspergillus flavus synergistically,whether EDTA is present at 1.0 mg/mL or 0.1 mg/mL (FIG. 7 and FIG. 8).This synergism extends to inhibition of Fusarium solani as well, as seenin FIG. 9. FIG. 10 and FIG. 11 show the synergistic inhibitory effect ofliposomal Amphotericin B and EDTA against A. fumigatus and F. solani.

[0090] The inventors have demonstrated, remarkably and for the firsttime, that the chelators and antifungal agents of the present inventionact together in a synergistic fashion to inhibit fungal pathogens. It iscontemplated that as a consequence of this synergism described abovebetween the chelators and the antifungal agents of the presentinvention, decreased dosages of antifungal agent will be sufficient toinduce a fungicidal effect in a patient with a fungal infection,relative to the dosage required when administering an antifungal agentalone. Advantageously, a decreased dosage of antifungal agent, when usedin conjunction with the chelators of the present invention, will serveto minimize any undesirable side effects which antifungal agents mayinduce in patients to whom they are administered.

[0091] Table 2 provides a representative list of antifungal agentsuseful in conjunction with the present invention. The list provided inTable 2 is not meant to be exhaustive. TABLE 2 ANTIFUNGAL AGENTS UK109,496 (Voriconazole) Terbinafine SCH 56592 BF-796 ER30346 MTCH 24 UK9746 BTG-137586 UK 9751 RMP-7/Amphotericin B T 8581 OmoconazoleFlutrimazole Amphotericin B Cilofungin LY121019 Nystatin LY303366(Echinocandin) Natamycin L-743872 (Pneumocandin) ClotrimazolePradimicins (MNS 18184) Miconazole Benanomicin Ketoconazole AmbisomeTerconazole ABLC Econazole Liposomal Amphotericin Itraconazole ABCDFluconazole Liposomal Nystatin Griseofulvin Nikkomycin Z Flucytosine

[0092] VI. Pharmaceutical Compositions and Routes of Administration

[0093] Pharmaceutical compositions of the instant invention comprise aneffective amount of at least a chelator dissolved or dispersed in apharmaceutically acceptable carrier, such as a pharmaceuticallyacceptable buffer, solvent or diluent, or aqueous medium. Pharmaceuticalcompositions of the instant invention may also comprise an effectiveamount of a chelator and an antifungal agent dissolved or dispersed in apharmaceutically acceptable carrier, such as a pharmaceuticallyacceptable buffer, solvent or diluent, or aqueous medium. Additionally,pharmaceutical compositions of the instant invention may comprise aneffective amount of a chelator operatively attached to a monoclonalantibody dissolved or dispersed in a pharmaceutically acceptablecarrier, such as a pharmaceutically acceptable buffer, solvent ordiluent, or aqueous medium. Also, pharmaceutical compositions of theinstant invention comprise an effective amount of a chelator operativelyattached to a monoclonal antibody, as well as an antifungal agent, alldissolved or dispersed in a pharmaceutically acceptable carrier, such asa pharmaceutically acceptable buffer, solvent or diluent, or aqueousmedium. Such compositions also can be referred to as inocula.

[0094] The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a human. As used herein the terms “pharmaceuticallyacceptable carrier” and “pharmaceutically acceptable buffer, solvent ordiluent” include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

[0095] The therapeutic compositions of the present invention may includeclassic pharmaceutical preparations. Administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,administration will be by orthotopic, intradermal subcutaneous,intramuscular, intraperitoneal or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients. For treatment of conditions ofthe lungs, the preferred route is aerosol delivery to the lung.

[0096] An effective amount of the therapeutic composition is determinedbased on the intended goal. The term “unit dose” or “dosage” refers tophysically discrete units suitable for use in a subject, each unitcontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses, discussed above, inassociation with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the protection desired.

[0097] Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting dose include physical and clinical state of thepatient, the route of administration, the intended goal of treatment(alleviation of symptoms versus cure) and the potency, stability andtoxicity of the particular therapeutic substance.

[0098] Additional formulations are suitable for oral administration.Oral formulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

[0099] As used herein the terms “contact”, “contacted”, and“contacting”, are used to describe the process by which an effectiveamount of a pharmacological agent, e.g., any of the compounds disclosedin the present invention, comes in direct juxtaposition with the targetcell.

[0100] For methods of treating mammals, pharmaceutical compositions maybe administered by a variety of techniques, such as parenteral, topicalor oral administration. For example, the compositions of the instantinvention may also be formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. The preparation of anaqueous composition that contains one of the inventive compounds as anactive ingredient will be known to those of skill in the art in light ofthe present disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for use in preparing solutions or suspensions upon the additionof a liquid prior to injection can also be employed; and thepreparations can also be emulsified.

[0101] Solutions of the inventive compositions as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

[0102] The pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions; examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oil andinjectable organic esters such as ethyloleate. Aqueous carriers includewater, alcoholic/aqueous solutions, saline solutions, parenteralvehicles such as sodium chloride, Ringer's dextrose, etc. Intravenousvehicles include fluid and nutrient replenishers. Preservatives includeantimicrobial agents, anti-oxidants, chelating agents and inert gases.The pH and exact concentration of the various components thepharmaceutical composition are adjusted according to well knownparameters. Sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions may also be useful. In allcases the form must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi.

[0103] The compositions of the instant invention may also be formulatedinto a composition in a neutral or salt form. Pharmaceuticallyacceptable salts include the acid addition salts (formed, e.g., with anyfree amino groups present), which are formed with inorganic acids suchas, for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric, mandelic, and the like. Salts formed withany free carboxyl groups can also be derived from inorganic bases suchas, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

[0104] The carrier can also be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0105] Sterile injectable solutions are prepared by incorporating thecompounds of the present invention in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

[0106] Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

[0107] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. In this connection, sterile aqueous media which can be employedwill be known to those of skill in the art in light of the presentdisclosure. For example, one dosage could be dissolved in 1 mL ofisotonic NaCl solution and either added to 1000 mL of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variations in dosage will necessarily occur dependingon the condition of the subject being treated. The person responsiblefor administration will, in any event, determine the appropriate dosefor the individual subject.

[0108] VII. Prevention and Treatment of Disseminated Fungal Infections

[0109] The pharmaceutical compositions of the invention will be providedto a patient having a fungal infection in an amount sufficient to exerta fungicidal or fungistatic effect upon fungi contacted by thecomposition. It will be understood with benefit of this disclosure thatsuch dosages may vary considerably according to the patient, theinfection presented by the patient, and the particular activeingredients comprising the pharmaceutical composition.

[0110] The antifungal agents of the present invention may beadministered to a patient in an amount ranging from about 0.001milligrams per kilogram of body weight per day to about 1000 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.001, 0.002,0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10,etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100, 101, 102, 103,104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800, 900, and about 1000mg per kg per day, and including all fractional dosages therebetween.

[0111] More preferably, the antifungal agents of the present inventionmay be administered to a patient in an amount ranging from about 0.01milligrams per kilogram of body weight per day to about 100 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.01, 0.02, 0.03,etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, andincluding all fractional dosages therebetween.

[0112] Most preferably, the antifungal agents of the present inventionmay be administered to a patient in an amount ranging from about 0.1milligrams per kilogram of body weight per day to about 10 mg per kg perday, including all intermediate dosages therebetween. It will be readilyunderstood that “intermediate dosages”, in these contexts, means anydosages between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,7, 8, 9 and about 10 mg per kg per day, and including all fractionaldosages therebetween.

[0113] The chelators of the present invention may be administered to apatient in an amount ranging from about 0.001 milligrams per kilogram ofbody weight per day to about 1000 mg per kg per day, including allintermediate dosages therebetween. It will be readily understood that“intermediate dosages”, in these contexts, means any dosages between thequoted ranges, such as about 0.001, 0.002, 0.003, etc.; 0.01, 0.02,0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2,1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.;50, 51, 52, 53, 54, etc.; 100, 101, 102, 103, 104, etc.; 500, 501, 502,503, etc.; 600, 700, 800, 900, and about 1000 mg per kg per day, andincluding all fractional dosages therebetween.

[0114] More preferably the chelators of the present invention may beadministered to a patient in an amount ranging from about 0.01milligrams per kilogram of body weight per day to about 100 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.01, 0.02, 0.03,etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12, 13, 14, etc.; 50, 51,52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mg per kg per day, andincluding all fractional dosages therebetween.

[0115] Most preferably the chelators of the present invention may beadministered to a patient in an amount ranging from about 0.1 milligramsper kilogram of body weight per day to about 10 mg per kg per day,including all intermediate dosages therebetween. It will be readilyunderstood that “intermediate dosages”, in these contexts, means anydosages between the quoted ranges, such as about 0.1. 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6,7, 8, 9, and about 10 mg per kg per day, and including all fractionaldosages therebetween.

[0116] The monoclonal antibodies operatively attached to chelators maybe administered to a patient in an amount ranging from about 0.001milligrams per kilogram of body weight per day to about 1000 mg per kgper day, including all intermediate dosages therebetween. It will bereadily understood that “intermediate dosages”, in these contexts, meansany dosages between the quoted ranges, such as about 0.001, 0.002,0.003, etc.; 0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10,etc.; 12, 13, 14, etc.; 50, 51, 52, 53, 54, etc.; 100, 101, 102, 103,104, etc.; 500, 501, 502, 503, etc.; 600, 700, 800, 900, and about 1000mg per kg per day, and including all fractional dosages therebetween.

[0117] More preferably, the monoclonal antibodies operatively attachedto chelators may be administered to a patient in an amount ranging fromabout 0.01 milligrams per kilogram of body weight per day to about 100mg per kg per day, including all intermediate dosages therebetween. Itwill be readily understood that “intermediate dosages”, in thesecontexts, means any dosages between the quoted ranges, such as about0.01, 0.02, 0.03, etc.; 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.; 3, 4, 5, 6, 7, 8, 9, 10, etc.; 12,13, 14, etc.; 50, 51, 52, 53, 54, etc.; 60, 70, 80, 90, and about 100 mgper kg per day, and including all fractional dosages therebetween.

[0118] Most preferably, the monoclonal antibodies operatively attachedto chelators may be administered to a patient in an amount ranging fromabout 0.1 milligrams per kilogram of body weight per day to about 10 mgper kg per day, including all intermediate dosages therebetween. It willbe readily understood that “intermediate dosages”, in these contexts,means any dosages between the quoted ranges, such as about 0.1. 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, etc.;3, 4, 5, 6, 7, 8, 9, and about 10 mg per kg per day, and including allfractional dosages therebetween.

[0119] The pharmaceutical compositions of the present invention may beadministered by any known route, including parenterally and otherwise.This includes oral, nasal (via nasal spray or nasal inhaler), buccal,rectal, vaginal or topical administration. Administration may also be byorthotopic, intradermal subcutaneous, intramuscular, intraperitoneal orintravenous injection and/or infusion. Such compositions may beadministered as pharmaceutically acceptable compositions that includepharmacologically acceptable carriers, buffers or other excipients. Thephrase “pharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to a human. For treatment of conditions ofthe lungs, the preferred route is aerosol delivery to the lung viabronchoalveolar lavage or the like.

[0120] VIII. Packaging and Kits

[0121] The container means of the kits will generally include at leastone vial, test tube, flask, bottle, syringe or other container means,into which the linked antibody/chelator may be placed, and preferably,suitably aliquoted. Where a second or third antifungal agent, otherchelator, or additional component is provided, the kit will alsogenerally contain a second, third or other additional container intowhich this component may be placed. The kits of the present inventionwill also typically include a means for containing theantibody/chelator, antifungal agent, other chelator, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

[0122] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLE 1 Synergy Study

[0123] The goal of this study was to determine if there is a synergisticeffect between EDTA and Amphotericin B and EDTA and Ambisomerespectively. The data collected are displayed in FIG. 5, FIG. 6, FIG.7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11. These figures are discussedelsewhere in the Detailed Description section. The studies wereconducted in a laboratory incubator at a constant temperature of 30° C.

[0124] The medium was a single lot of liquid RPMI 1640 medium (WhittakerBioproducts, Inc., Walkersville, Md.) supplemented with 0.3 g ofL-glutamine per liter and 0.165 M MOPS buffer (34.54 g/liter) andwithout sodium bicarbonate.

[0125] Test inocula contained approximately 1×10³ to 1×10⁴ conidia/mL.To induce conidium and sporangiophore formation, fungi were grown onsabouraud dextrose agar plates at 35° C. for 5 to 7 days. Each funguswas then covered with approximately 2 mL sterile 0.85% saline water. Thesuspension was then harvested by gently probing the colonies withsterile glass rods. The resulting mixture of conidia or sporangiophoresand hyphal fragments was withdrawn and filtered through a sterile 4×4gauze to a sterile tube. The homogenous suspension was later mixed witha vortex mixer for 30 s and the densities of the suspension were readand adjusted to a range of 80 to 85% transmittance. Inoculum of 0.1 mLwas delivered to each flask containing 20 mL of RPMI and drug dilutionseries. The final conidia concentration ranged from 1×10³ to 1×10⁴conidia/mL. A control flask was maintained without any drugs. The flaskswere incubated in a shaker at 30° C. for 24 to 48 h. Glass beads wereadded to all flasks with visible fungal growth in an attempt tohomogenize the solution and achieve even distribution of conidia forculture. Cultures were done at 0, 4, 24, and 48 h on sabouraud dextroseagar plates and incubated at 35° C. for 48 h.

[0126] Amphotericin B for injection, USP (Gensia Laboratories, LTD.) wassuspended and diluted in sterile water and stored at 1 mg/mL in a glassvile in the dark at −70° C.

[0127] Ambisome was obtained in 50 mg vials and used immediately uponopening of the vial. Typically, 50 mg of Ambisome was diluted in 12 mLof sterile water. Further dilutions were performed as needed.

[0128] Edetate disodium INJ., USP (Abbott Laboratories, North Chicago,Ill.) was stored at a concentration of 150 mg/mL at 4° C.

[0129] Further dilutions were made to achieve the desired concentrationof each drug at the time of the study. For Amphotericin B and Ambisome,the concentration was 1.0 μg/mL and for EDTA the concentrations were 0.1and 1.0 mg/mL.

EXAMPLE 2 Inhibition Study

[0130] The goal of this study was to determine if chelators have aninhibitory effect on species of Aspergillus, Fusarium, and Candida. Thedata collected are displayed in FIG. 1, FIG. 2, FIG. 3 and FIG. 4. Thesefigures are discussed elsewhere in the Detailed Description section. Aspectrophotometer was used at a frequency of 660 nm to measure theabsorbency of the solution.

[0131] For molds, all inocula were started at 1×10⁴ conidia/mL. Foryeast and bacteria, all inocula were started at 1×10⁶ cfu/mL. The mediumused was Mueller-Hinton.

[0132] The concentration of chelator in these studies was 0.35 mg EDTAper mL water.

EXAMPLE 3 In Vivo Prophetic Model

[0133] In vivo studies will be conducted with either rabbits or mice,both of which are suitable animal models. Immunosupression withcyclophosphamide should be given intravenously 3 days prior tocommencement of the study in order to achieve neutropenia by the day ofanimal infection.

[0134] Treatment with all drugs begins 18 to 24 h after infection andcontinues for 10 days.

[0135] All animals surviving to day 11 are sacrificed. Their lungs,kidneys, liver, and spleen are removed and transferred into 5 mL ofsterile saline, homogenized in a tissue grinder for 15 to 30 sec, anddiluted to 10⁻¹, 10⁻², 10⁻³, and 10⁻⁴. A total of 1.0 mL of eachdilution is spread onto a sabouraud dextrose agar plate and allowed togrow by incubating them at 37° C. The plates are then counted forquantitative analysis. Also, histopathology analysis will be conductedon all organs analyzed.

[0136] All of the compositions and methods disclosed and claimed hereincan be made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

[0137] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0138] Benisek and Richards, J. Biol. Chem., 243:4267, 1968.

[0139] Campbell, In: Monoclonal Antibody Technology, LaboratoryTechniques in Biochemistry and Molecular Biology, Vol. 13, Burden andVon Knippenberg, (Eds.), Amsterdam, Elseview,pp 75-83, 1984

[0140] DeRiemer, Meares, Goodwin, Diamanti, J. Med Chem., 22:019, 1979.

[0141] Gefter et al., Somatic Cell Genet., 3:231-236, 1977.

[0142] Gelewitz, Riedemann, Klotz, Arch. Biochem. Biophys., 53:411,1954.

[0143] Goding, 1986, In: Monoclonal Antibodies: Principles and Practice,2nd ed., Academic Press, Orlando, Fla., pp. 60-61, 65-66, 71-74, 1986.

[0144] Goodwin, Sundberg, Diamanti, Meares, In: Radiopharmaceuticals,Society of Nuclear Medicine, New York, p. 80, 1975.

[0145] Hnatowich, Layne, Childs, Lanteigne, Davis, Griffin, Doherty,Science, 220:613, 1983.

[0146] Khaw, Fallon, Strauss, Haber, Science, 209:295, 1980.

[0147] Kohler and Milstein, Nature, 256:495-497, 1975.

[0148] Krejcarek and Tucker, “Covalent attachment of chelating groups tomacromolecules,” Biochem. Biophys. Res. Commun., 77:581-585, 1977.

[0149] Leung, Meares, Goodwin, Int. J. Appl. Radiot. Isot., 29:687,1978.

[0150] Meares, Goodwin, Leung, Girgis, Silvester, Nunn, Lavender,“Covalent attachment of metal chelates to proteins: The stability invivo and in vitro of the conjugate of albumin with a chelate of¹¹¹indium,” Proc. Natl. Acad. Sci. U.S.A., 73:3803-3806, 1976.

[0151] Meares and Wensel, Accts. Chem. Res., 17:202, 1984.

[0152] Sokolovsky, Riordan, Vallee, Biochem. Biophys. Res. Commun.,27:20, 1967.

[0153] Sundberg, Meares, Goodwin, Diamanti, Nature, 250:587; J. Med.Chem., 17:1304, 1974.

[0154] Yeh, Meares, Goodwin, J. Radioanal. Chem., 53:327, 1979.

[0155] Yeh, Sherman, Meares, “A new route to “bifunctional” chelatingagents: conversion of amino adds to analogs ofethylenedinitrilotetraacetic acid,” Anal. Biochem., 100:152-159, 1979.

[0156] U.S. Pat. No. 5,362,754 by Raad et al.

[0157] U.S. patent application Ser. No. 08/317,309 by Raad et al.

What is claimed is:
 1. A method of treating a systemic fungal infectioncomprising: (a) obtaining a therapeutically effective amount of apharmaceutical composition comprising at least one chelator, at leastone antifungal agent and a pharmaceutical excipient, diluent oradjuvant; and (b) administering said pharmaceutical composition to apatient having a fungal infection.
 2. The method of claim 1, whereinsaid chelator in said pharmaceutical composition may be selected fromthe group of chelators in Table
 1. 3. The method of claim 2, whereinsaid chelator is EDTA.
 4. The method of claim 1, wherein said antifungalagent may be selected from the group of antifungal agents in Table
 2. 5.The method of claim 4, wherein said antifungal agent is Amphotericin B.6. The method of claim 1, wherein said pharmaceutical compositioncomprises about 0.001 mg to about 1000 mg of chelator.
 7. The method ofclaim 1, wherein said pharmaceutical composition comprises about 0.001mg to about 1000 mg of antifungal agent.
 8. The method of claim 1,wherein said pharmaceutical composition may be administered byinjection, bronchoalveoloar lavage, or by nasal drops, nasal spray, ornasal inhaler.
 9. The method of claim 1, wherein said pharmaceuticalcomposition may be administered by injection.
 10. A pharmaceuticalcomposition comprising at least one antifungal agent and at least onechelator.
 11. The pharmaceutical composition of claim 10, wherein thechelator may be selected from the group of chelators in Table
 1. 12. Thepharmaceutical composition of claim 10, wherein the antifungal agent maybe selected from the group of antifungal agents in Table
 2. 13. Thepharmaceutical composition of claim 11, wherein the chelator is EDTA.14. The pharmaceutical composition of claim 12, wherein the antifungalagent is Amphotericin B.
 15. The pharmaceutical composition of claim 10,wherein the chelator is EDTA and the antifungal agent is Amphotericin B.16. The pharmaceutical composition of claim 10, further defined ascomprising about 0.001 mg to about 1000 mg of chelator.
 17. Thepharmaceutical composition of claim 10, further defined as comprisingabout 0.001 mg to about 1000 mg of antifungal agent.
 18. Thepharmaceutical composition of claim 10, further comprising at least onemonoclonal antibody specific for a targeted species of fungus.
 19. Thepharmaceutical composition of claim 10, wherein said monoclonal antibodyis operatively attached to said chelator.
 20. A pharmaceuticalcomposition comprising at least one chelator, at least one antifungalagent and at least one monoclonal antibody, wherein said monoclonalantibody is operatively attached to said chelator.
 21. Thepharmaceutical composition of claim 20, wherein the chelator may beselected from the group of chelators in Table
 1. 22. The pharmaceuticalcomposition of claim 20, wherein the antifungal agent may be selectedfrom the group of antifungal agents in Table
 2. 23. The pharmaceuticalcomposition of claim 21, wherein the chelator is EDTA.
 24. Thepharmaceutical composition of claim 22, wherein the antifungal agent isAmphotericin B.
 25. The pharmaceutical composition of claim 20, whereinthe chelator is EDTA and the antifungal agent is Amphotericin B.
 26. Thepharmaceutical composition of claim 20, further defined as comprisingabout 0.001 mg to about 1000 mg of chelator.
 27. The pharmaceuticalcomposition of claim 20, further defined as comprising about 0.001 mg toabout 1000 mg of antifungal agent.
 28. A method for treating a systemicfungal infection comprising administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition comprising at least onechelator, at least one antifungal agent and at least one monoclonalantibody, wherein said monoclonal antibody is operatively attached tosaid chelator.